Blood cells are derived from progenitor cells which are derived from hematopoietic stem cells. The process whereby it is determined whether a stem cell will ultimately differentiate to form erythrocytes or leukocytes is a complex and not well understood process thought to be controlled by hormonal signals received by the cells during maturation. Although the precise scheme of hematopoiesis is not presently understood, the role of some hematopoietically-active agents is.
The process by which stem cells are induced to become mature red blood cells, erythropoiesis, is under the control of erythropoietin, a glycoprotein hormone synthesized primarily in the kidneys. Erythropoietin, which itself is regulated by the number of circulating erythrocytes, induces progenitor cells to become committed to differentiate into erythrocytes.
Several mammalian cell lines serve as models for studying erythropoiesis. These cell culture models differ as to the species from which they were derived (e.g., mouse, human, etc.) and as to their relative stage of erythrodifferentiation. Both the species of origin and the stage of maturation of a cell line are important parameters which must be considered when studying erythropoiesis. Many biologically-active molecules involved in the process of red cell differentiation have a marked effect in one species, but no activity in another. Also, proteins which clearly affect erythrodifferentiation at one stage of development may have no effect at a later or earlier stage. Thus, narrow specificities in the activity of molecules active in erythrodifferentiation are commonly seen.
The K562 cell line is a human cell line characteristic of cells at a relatively mature stage in erythrodifferentiation. It expresses such red cell markers as glycophorin A, spectrin and antigen i. K562 cells undergo differentiation and accumulate hemoglobin (one of several heme proteins, and the essential one for binding and transporting oxygen) upon induction by hemin or other chemical inducers.
A cell line that has been used to study erythrodifferentiation in mice is the mouse Friend leukemia cell line. It is a model for differentiation established with Friend virus. Several chemical inducers that work in this mouse cell line do not work with human cell lines. Furthermore, the presently known chemical inducers for these cell lines are not the physiological regulators for erythropoiesis in vivo.
Therefore, results derived from studies conducted on certain cell lines do not assure that the same biological agent will work in an in vivo system or in cells which may be at a different stage of development and/or which may be derived from a different species.
In this respect, bone marrow is a significant source of progenitor cells for the study of erythrodifferentiation. Progenitor cells cultured from bone marrow respond to many physiological hematopoietic factors found in vivo.
A preparation of placental extract was shown by Ikawa et al., Gann. 66, 583-584 (1975) to enhance .delta.-aminolevulinic acid synthetase and the percentage of benzidine staining cells (i.e., heme-containing cells) in a mouse Friend leukemia cell culture. Recently, using the same mouse cell line, Eto, et al., Biochem. Biophys. Res. Comm. 142, 1095-1103 (1987), reported that EDF (erythroid differentiating factor) increased the percentage of cells staining for an unidentified heme protein, and that EDF and porcine FRP had similar amino-termini.
Inhibin, a heterodimeric protein consisting of an .alpha.-subunit (18,000 daltons) and one of two B subunits (.beta..sub.A or .beta..sub.B) (14,000 daltons), and FRP, a homodimer consisting of a pair of B subunits, are functionally antagonistic, hypophysiotropic hormones which were shown by Vale, et al., Nature 321, 776-779 (1986) to respectively suppress and enhance the secretion of follicle-stimulating hormone (FSH).
We have found, unexpectedly, that FRP and inhibin exert a novel and complex humoral regulatory control over erythropoiesis in humans. FRP induces hemoglobin accumulation in K562 cells, and inhibin functionally antagonizes this effect. In addition to the regulatory effect on hemoglobin accumulation, FRP and inhibin have profound augmenting (FRP) and suppressing (inhibin) activity on erythropoietin-induced proliferation and differentiation of bone marrow progenitor cells.